Basement membranes are a highly specialized part of the extracellular matrix and they form the thin sheets that separate the cells of organ tissues from the fibrillar connective tissues. The basement membranes form a substratum for orderly growing cells in the body and they play an important role in cell differentiation during the formation of organs in the developing embryo. In addition, they also play a key role in the correct regeneration of tissues following injuries such as during postwound reformation of skin and growth of nerves.
Furthermore, the basement membranes also have crucial special functions, such as in the filtration of macromolecules in kidney and blood vessels. This is best exemplified by the renal glomerular basement membrane where the filtration of blood results in the formation of primary urine that is devoid of cells and large macromolecules (proteins).
The basement membranes are composed of several proteins, many of which are found only in the membranes. Type IV collagen is the major structural component but other specific protein components include laminin, entactin (nidogen) and a heparin sulfate proteoglycan. Additionally, the basement membranes may contain fibronectin and type VII collagen that are also present in other tissues. The differences in the molecular composition of basement membrane in different tissues is not well known but a protein called pemphigoid antigen is probably only present in the basement membrane of skin. It is currently thought that there exists a number of other proteins that are specific for certain basement membranes.
Type IV collagen is the predominant structural component of basement membranes and it can provide up to 60% of the structure. As it is true for collagens in general, type IV collagen molecules are composed of three .alpha. chains that are coiled around each other to form a rod-like triple helical molecule that is about 1.5 nm in diameter and about 400 nm in length. At the carboxy terminal end the molecule has a large globular noncollagenous domain, called NC-domain, that has a diameter of about 15 nm. Single type IV collagen molecules are linked with each other into a complex, flexible network-like structure (Timpl, Eur.J.Biochem., 180, 487-502, 1989) into which other basement membrane components are bound.
It was previously thought that the major form of type IV collagen was composed of two kinds of chains, .alpha.1(IV) and .alpha.2(IV), with the molecular formula [.alpha.1(IV)].sub.2 .alpha.2(IV). Along this line, the applicants have determined the complete amino acid sequence of both the human .alpha.1(IV) and the .alpha.2(IV) chains from cloned cDNA molecules (Soininen, et al., FEBS Lett., 225, 188-194, 1987; Hostikka and Tryggvason, J.Biol.Chem., 263, 19488-19493, 1988). The results showed that the .alpha.1(IV) chain contains 1,642 amino acid residues as compared with 1,676 residues for the .alpha.2(IV) chain. The carboxy terminal NC-domains of both chains are very similar with 63% identical amino acid residues. The sequence homology of the two chains in the triple helical region is considerably less or 49%.
However, recently the existence of two additional .alpha. chains, termed .alpha.3(IV) and .alpha.4(IV) have also been reported to be present in the basement membrane of lens (Butkowski, et al., J.Biol.Chem., 262, 7874-7877, 1987). Therefore, the currently available data indicates that there are type IV collagen molecules with varying chain compositions and the different forms may contribute to functional differences of the basement membrane in various tissues The applicants' present discovery of a new type IV collagen .alpha. chain, termed .alpha.5(IV), demonstrates that type IV collagen composition and therefore the whole basement membrane matrix is even more complex than was previously thought.
Due to the wide distribution of basement membranes in the body, they are frequently affected in local and systemic diseases and in many instances, the consequent pathological changes lead to severe clinical complications. These disease may be both genetically determined inherited diseases that are due to gene mutations leading to an abnormal function of the basement membrane or they can be acquired, i.e. complications of diseases that do not primarily involve the basement membrane. Examples of inherited diseases are: (1) the congenital nephrotic syndrome that is characterized by extensive leak of blood proteins through the renal glomerular basement membrane into urine (proteinuria); and, (2) the Alport's syndrome where malfunction of the basement membranes leads to the passage of blood cells into urine (hematuria), eye lesions and hearing loss. The actual gene defects leading to the congenital nephrotic syndrome or the Alport's syndrome are yet completely unknown. Both diseases are lethal but they may be treated with kidney transplantation.
The best known example of an acquired basement membrane disease is diabetes mellitus where the basement membrane structure is affected in almost all tissues of the body resulting in dysfunction of small blood vessels (microangiopathy), kidneys (nephropathy), and nerves (neuropathy). The biochemical alterations leading to these malfunctions are still poorly understood.
The present invention, directed to a process for isolating and identifying the nucleotide sequence of a new polypeptide chain of human type IV collagen (i.e. .alpha.5(IV)), is of important significance in the diagnosis of basement membrane disorders. In this regard, genetic mutations leading to the abnormal function of the basement membrane can be detected in the DNA of affected individuals by comparing the nucleotide sequences, etc.